Adeno-associated virus (AAV) is a replication-deficient human parvovirus, the genome of which is about 4.6 kb in length, including 145 nucleotide inverted terminal repeats (ITRs). Two open reading frames encode a series of rep and cap polypeptides. Rep polypeptides (rep78, rep68, rep62 and rep40) are involved in replication, rescue and integration of the AAV genome. The cap proteins (VP1, VP2 and VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5′ and 3′ ends are 145 bp inverted terminal repeats (ITRs), the first 125 bp of which are capable of forming Y- or T-shaped duplex structures. Of importance for the development of AAV vectors, the entire rep and cap domains can be excised and replaced with a therapeutic or reporter transgene [B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp.155–168 (1990)]. It has been shown that the ITRs represent the minimal sequence required for rescue, packaging, and integration of the AAV genome.
The wild type, nonpathogenic human virus is capable of infecting a wide variety of cells and establishing a latent infection of the cell via a provirus that integrates at high frequency into a specific region of chromosome 19 [Kotin, R. M. et al, Proc. Natl. Acad. Sci. USA 87, 2211–2215 (1990); Samulski, R. J. et al. EMBO J. 10, 3941–3950 (1991)]. Production of infectious virus and replication of the virus does not occur unless the cell is coinfected with a helper virus, such as adenovirus or herpesvirus. Upon infection with a helper virus, the genes of latent AAV (i.e., rep and cap) are activated, resulting in rescue of the AAV provirus, replication of the AAV genome, and formation of AAV virions, as well as generation of additional helper virus. The infecting parental ssDNA is expanded to duplex replicating form (RF) DNAs in a rep dependent manner. The rescued AAV genomes are packaged into preformed protein capsids (icosahedral symmetry approximately 20 nm in diameter) and released as infectious virions that have packaged either + or − ss DNA genomes following cell lysis.
AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells. Progress towards establishing AAV as a transducing vector for gene therapy has been slow. Evaluation of recombinant AAV (rAAV) produced recombinantly and exclusive of wildtype AAV reformed by recombinant methods in preclinical models of gene therapy has been limited for a variety of reasons, primarily because methods of production are inefficient and often generate substantial quantities of replication competent AAV. Replication defective forms of AAV are created by transfecting vector DNA (transgene flanked by AAV ITRs) together with a rep/cap expressing plasmid concurrent with adenovirus infection [Samulski, R. et al, J. Virol, 61(10):3096–3101 (1987)] or transfection with an adenovirus helper plasmid [Ferrari, F. K. et al, Nature Med., 3, 1295–1297 (1997)]. The standard method, based on transient transfection, is not easily scaled-up, making it difficult to obtain virus [Fisher, K. J. et al. J. Virol. 70, 520–532 (1996)]. Furthermore, preparations are invariably contaminated with replication competent AAV (rcAAV) formed by, for example, nonhomologous recombination, during the process of transfection. Another method for producing rAAV has been described based on the simultaneous transient transfection of cis and trans plasmids together with an adenovirus helper plasmid [Ferrari et al, Nature Med. 3: 1295–1297 (1997)]. This approach has the advantage of minimizing contaminating adenovirus but suffers from the creation of rcAAV and difficulties in scaling-up. A specific obstacle to the use of AAV for delivery of DNA, especially for therapeutic applications, has been lack of highly efficient schemes for encapsidation of recombinant genomes and production of infectious virions [R. Kotin, Hum. Gene Ther., 5:793–801 (1994)].
Problems exist in attempts to improve AAV production including the use of cell lines stably expressing vector and/or rep/cap. Creation of a cell line stably expressing rep and cap has been difficult because of cellular toxicity of rep gene products. Several cell lines previously described do not express sufficient quantities of rep or cap to sustain high titer production of vector [Tamayose, K. et al, Hum. Gene Thera. 7, 507–513 (1996); Yang, Q., et al, J. Virol. 68, 4847–4856 (1994); see, also, Clark, K. R. et al, Hum. Gene Thera. 6, 1329–1341 (1995) and U.S. Pat. No. 5,658,785]. Other strategies have been described that provide more efficient ways to transiently express and regulate rep and cap [Mamounas, M., et al, Gene Thera. 2, 429–432 (1995); and Flotte, T. R. et al. Gene Thera. 2, 29–37 (1995)].
Disadvantages of current methods for production of rAAV that employ transfection of rAAV genome into host cells followed by co-infection with wild-type AAV and adenovirus, include the production of unacceptably high levels of wild-type AAV, little recombinant gene expression and inefficient integration. Another recognized means for manufacturing transducing AAV virions entails co-transfection with two different, yet complementing plasmids. One of these plasmids contains a therapeutic or reporter transgene flanked (sandwiched) between the two cis acting AAV ITRs. The AAV components that are needed for rescue and subsequent packaging of progeny recombinant genomes are provided in trans by a second plasmid encoding the viral open reading frames for rep and cap proteins. However, both rep and cap are toxic to the host cells. This toxicity has been the major source of difficulty in providing these genes in trans for the construction of a useful rAAV gene therapy vector.
There remains a need in the art for additional methods permitting the efficient production of AAV and recombinant AAV viruses for use as vectors for somatic gene therapy at high titers not previously achieved.